Furin-knockdown bi-functional RNA

ABSTRACT

Compositions and methods to attenuate the immunosuppressive activity of TGF-β through the use of bi-functional shRNAs was substituted therefor described herein. The bi-functional shRNAs of the present invention knocks down the expression of furin in cancer cells to augment tumor antigen expression, presentation, and processing through expression of the GM-CSF transgene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/289,661, filed Dec. 23, 2009, the entire contents of which isincorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of vaccinedevelopment, and more particularly, to the development of compositionsand methods for making and using an autologous cancer vaccinegenetically modified for Furin knockdown and GM-CSF expression.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

REFERENCE TO A SEQUENCE LISTING

The present application includes a Sequence Listing filed separately inan electronic format as required by 37 C.F.R §1.821-1.825.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with the development of genetically modified whole cellcancer vaccines. More specifically, the present invention relates tovaccines capable of augmenting tumor antigen expression, presentation,and processing through expression of the GM-CSF transgene andattenuating secretory immunosuppressive activity of TGF-β via furinbi-functional shRNA transgene induced knockdown.

The prevailing hypothesis for immune tolerance to cancer vaccinesinclude the low immunogenicity of the tumor cells, the lack ofappropriate presentation by professional antigen presenting cells,immune selection of antigen-loss variants, tumor inducedimmunosuppression, and tumor induced privileged site. Whole cancer cellvaccines can potentially solicit broad-based, polyvalent immuneresponses to both defined and undefined tumor antigens, therebyaddressing the possibility of tumor resistance through downregulationand/or selection for antigen-loss variants. An example of a method formaking a master cell bank of whole cell vaccines for the treatment ofcancer can be found in U.S. Pat. No. 7,763,461 issued to Link et al.(2010). According to the '461 patent tumor cells are engineered toexpress an α (1,3) galactosyl epitope through ex-vivo gene therapyprotocols. The cells are then irradiated or otherwise killed andadministered to a patient. The a galactosyl epitope causes opsonizationof the tumor cell enhancing uptake of the opsonized tumor cell byantigen presenting cells which results in enhanced tumor specificantigen presentation. The animal's immune system thus is stimulated toproduce tumor specific cytotoxic cells and antibodies which will attackand kill tumor cells present in the animal

Granulocyte-macrophage colony-stimulating factor, often abbreviated toGM-CSF, is a protein secreted by macrophages, T cells, mast cells,endothelial cells and fibroblasts. When integrated as a cytokinetransgene, GM-CSF enhances presentation of cancer vaccine peptides,tumor cell lysates, or whole tumor cells from either autologous orestablished allogeneic tumor cell lines. GM-CSF induces thedifferentiation of hematopoietic precursors and attracts them to thesite of vaccination. GM-CSF also functions as an adjuvant for dendriticcell maturation and activational processes. However, GM-CSF-mediatedimmunosensitization can be suppressed by tumor produced and/or secreteddifferent isoforms of transforming growth factor beta (TGF-β). The TGF-βfamily of multifunctional proteins possesses well knownimmunosuppressive activities. The three known TGF-β ligands (TGF-β1, β2,and β3) are ubiquitous in human cancers. TGF-β overexpression correlateswith tumor progression and poor prognosis. Elevated TGF-β levels withinthe tumor microenvironment are linked to an anergic antitumor response.TGF-β inhibits GM-CSF induced maturation of dendritic cells and theirexpression of MHC class II and co-stimulatory molecules. This negativeimpact of TGF-β on GM-CSF-mediated immune activation supports therationale of depleting TGF-β secretion in GM-CSF-based cancer cellvaccines.

All mature isoforms of TGF-β require furin-mediated limited proteolyticcleavage for proper activity. Furin, a calcium-dependent serineendoprotease, is a member of the subtilisin-like proprotein convertasefamily. Furin is best known for the functional activation of TGF-β withcorresponding immunoregulatory ramifications. Apart from the previouslydescribed immunosuppressive activities of tumor secreted TGF-β,conditional deletion of endogenously expressed furin in T lymphocyteshas been found to allow for normal T-cell development, but impairedfunction of regulatory and effector T cells, which produced less TGF-β1.Furin expression by T cells appears to be indispensable in maintainingperipheral tolerance, which is due, at least in part, to itsnon-redundant, essential function in regulating TGF-β1 production.

High levels of furin have been demonstrated in virtually all cancerlines. The inventors and others have found that up to a 10-fold higherlevel of TGF-β1 may be produced by human colorectal, lung cancer, andmelanoma cells, and likely impact the immune tolerance state by a highermagnitude. The presence of furin in tumor cells likely contributessignificantly to the maintenance of tumor directed TGF-β peripheralimmune tolerance. Hence furin knockdown (via RNA interference mechanism)represents a novel and attractive approach for optimizingGM-CSF-mediated immunosensitization. Vaccines based on the phenomenon ofRNA interference (RNAi) have been previously described, for e.g. U.S.Patent Application No. 20040242518 (Chen et al. 2004) provides methodsand compositions for inhibiting influenza infection and/or replicationbased on the phenomenon of RNAi as well as systems for identifyingeffective siRNAs and shRNAs for inhibiting influenza virus and systemsfor studying influenza virus infective mechanisms. The invention alsoprovides methods and compositions for inhibiting infection,pathogenicity and/or replication of other infectious agents,particularly those that infect cells that are directly accessible fromoutside the body, e.g., skin cells or mucosal cells. In addition, theinvention provides compositions comprising an RNAi-inducing entity,e.g., an siRNA, shRNA, or RNAi-inducing vector targeted to an influenzavirus transcript and any of a variety of delivery agents. The inventionfurther includes methods of use of the compositions for treatment ofinfluenza

Interferon-gamma (γIFN) is a key immunoregulatory cytokine that plays acritical role in the host innate and adaptive immune response and intumor control. Also known as type II interferon, γIFN is a single-copygene whose expression is regulated at multiple levels. γIFN coordinatesa diverse array of cellular programs through transcriptional regulationof immunologically relevant genes. Initially, it was believed that CD4+T helper cell type 1 (Th1) lymphocytes, CD8+ cytotoxic lymphocytes, andNK cells exclusively produced γIFN. However, there is now evidence thatother cells, such as B cells, NKT cells, and professionalantigen-presenting cells (APCs) secrete γIFN. γIFN production byprofessional APCs [monocyte/macrophage, dendritic cells (DCs)] actinglocally may be important in cell self-activation and activation ofnearby cells. γIFN secretion by NK cells and possibly professional APCsis likely to be important in early host defense against infection,whereas T lymphocytes become the major source of γIFN in the adaptiveimmune response. Furthermore, a role for γIFN in preventing developmentof primary and transplanted tumors has been identified. γIFN productionis controlled by cytokines secreted by APCs, most notably interleukin(IL)-12 and IL-18. Negative regulators of γIFN production include IL-4,IL-10, glucocorticoids, and TGF-β.

SUMMARY OF THE INVENTION

The present invention includes a unique method of inhibiting TGF-βthrough RNA interference with furin, a proprotein convertase involvedcritically in the functional processing of all TGF-β isoforms. The FANGvector uniquely incorporates a bi-functional small hairpin construct(shRNA^(furin)) specific for the knockdown of furin. The bi-functionalshRNA^(furin) of the present invention comprises a two stem-loopstructures with a miR-30a backbone. The first stem-loop structure is thesiRNA component, while the second stem-loop structure is the miRNA-likecomponent. The strategy is to use a single targeted site for bothcleavage and sequestering mechanisms of RNA interference. The FANGconstruct contains GM-CSF and the bi-functional shRNA^(furin)transcripts under the control of a mammalian promoter (CMV) that drivesthe entire cassette. This construct is used to generate an autologous(i.e., patient specific) cancer vaccine genetically modified for furinknockdown and GM-CSF expression.

The construct used to produce the FANG vaccine in the present inventionincludes a bi-functional shRNA^(furin)/GMCSF expression vector plasmidcomprising two nucleic acid inserts. The first nucleic acid insert isoperably linked to a promoter, and it encodes a Granulocyte MacrophageColony Stimulating Factor (GM-CSF) cDNA. The second nucleic acid insertis also operably linked to the promoter, and it encodes one or moreshort hairpin RNAs (shRNA) capable of hybridizing to a region of an mRNAtranscript encoding furin, thereby inhibiting furin expression via RNAinterference. The bi-functional shRNA of the present invention has twomechanistic pathways of action, that of the siRNA and that of the miRNA.Thus, the bi-functional shRNA of the present invention is different froma traditional shRNA, i.e., a DNA transcription derived RNA acting by thesiRNA mechanism of action or from a “doublet shRNA” that refers to twoshRNAs, each acting against the expression of two different genes but inthe traditional siRNA mode.

In one embodiment of the invention, the GM-CSF is human. The shRNA isbi-functional, incorporating both siRNA (cleavage dependent) andmiRNA-like (cleavage-independent) motifs simultaneously. In oneembodiment of the present invention, the shRNA is both the cleavagedependent and cleavage independent inhibitor of furin expression. Theexpression vector may contain a picornaviral 2A ribosomal skip peptideintercalated between the first and the second nucleic acid inserts, andthe promoter may be CMV mammalian promoter which could contain a CMV IE5′ UTR enhancer sequence and a CMV IE Intron A. The mRNA sequencestargeted by the bi-functional shRNA are not limited to the coding regionof the furin mRNA transcript; in one embodiment, the shRNA may targetthe 3′ untranslated region (UTR) sequence of the furin mRNA transcript.

The present invention also includes a vector that may be used tospecifically knock down the expression of furin in target cells. ThisshRNA^(furin) expression vector comprises a nucleic acid insert operablylinked to a promoter. Such insert encodes one or more short hairpin RNAs(shRNA) capable of hybridizing to a region of an mRNA transcriptencoding furin, thereby inhibiting furin expression via RNAinterference. The bi-functional shRNA may simultaneously incorporatesiRNA (cleavage dependent) and miRNA (cleavage-independent) motifs, andinhibit furin expression in both a cleavage dependent and cleavageindependent manner. Additionally, the expression vector may target thecoding region of the furin mRNA transcript, or in the alternative it maytarget the 3′ UTR region sequence of the furin mRNA transcript.

The present invention further provides a composition comprising atherapeutically effective amount of cells with an expression vector. Theexpression of the composition comprises a cell transfected with a firstnucleic acid insert operably linked to a promoter, wherein the firstinsert encodes GM-CSF and a second nucleic acid insert operably linkedto the promoter, wherein the second insert encodes one or more shorthairpin RNAs (shRNA) capable of hybridizing to a region of a mRNAtranscript encoding furin, thereby inhibiting furin expression via RNAinterference. In one aspect the GM-CSF is human. The encoded shRNAincorporates siRNA (cleavage dependent) and miRNA (cleavage-independent)motifs and functions as both a cleavage dependent and cleavageindependent inhibitor of furin expression. The shRNA is further definedas a bi-functional shRNA.

A picornaviral 2A ribosomal skip peptide is intercalated between thefirst and the second nucleic acid inserts of the promoter, wherein thepromoter is a CMV mammalian promoter, enhancer, and intron. The regiontargeted by the shRNA of the present invention is the 3′ UTR regionsequence of the furin mRNA transcript. Alternatively, the shRNA can alsotarget the coding region of the furin mRNA transcript. As per thepresent invention the cells are autologous tumor cells, xenograftexpanded autologous tumor cells or allogeneic tumor cells. In specificaspects of the present invention the cells are xenograft expandedallogeneic tumor cells and comprises 1×10⁷ cells to 2.5×10⁷ cells. Thecomposition described herein further comprises a therapeuticallyeffective dose of γIFN (gamma interferon), wherein the therapeuticallyeffective dose of γIFN is 50 or 100 μg/m².

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A-1C show an assessment of GMCSF expression and TGF-β1 and -β2knockdown, summarizing (FIG. 1A) TGF-β1, (FIG. 1B) TGF-β2, and (FIG. 1C)GM-CSF protein production before and after FANG or TAG (TAG 004) plasmidtransfection. Values represent ELISA determinations of cytokineproduction in harvested autologous cancer cells transfected with FANG.Data represents autologus vaccines independently generated from 9patients who underwent FANG processing (FANG 001-009). One patient hadsufficient tissue to construct both a FANG (blue) and TAG vaccine (red)(FANG 004/TAG 004);

FIG. 2 shows the siRNA targeted regions of furin mRNA. Prospective siRNAtargeting regions in 3′-UTR and encoding regions of furin mRNA and thetargeted sequence by each siRNA;

FIG. 3 shows the overall survival for Cohort 1 versus Cohorts 2 and 3for advanced-stage patients (n=61; P=0.0186);

FIG. 4 shows a schematic diagram of GM-CSF-TGF-β2 antisense plasmid;

FIG. 5 shows the expression of GM-CSF in NCI-H-460 Squamous Cell andNCI-H-520, Large Cell (NSCLC) containing the pUMVC3-GM-CSF-2A-TGF-β2antisense vector, in vitro;

FIG. 6 shows that TGF-β2 levels are reduced in NCI-H-460 Squamous Celland NCI-H-520, Large Cell (NSCLC) with the pUMVC3-GM-CSF-2A-TGF-β2antisense vector;

FIG. 7 shows that a 251 base pair probe specifically detects theGM-CSF-2A-TGF-β2 transcript expressed in vitro in NCI-H-460 andNCI-H-520 cells (lanes 6 and 10);

FIG. 8 shows the GM-CSF expression in TAG vaccines;

FIG. 9 shows the TGF-β2 expression in TAG vaccines;

FIGS. 10A and 10B show expression of (FIG. 10A) TGF-β1 and (FIG. 10B)TGF-β2 in human cancer lines following siRNA furin knockdown; and

FIG. 11 shows the plasmid construct of FANG.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein the term “nucleic acid” or “nucleic acid molecule” refersto polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), oligonucleotides, fragments generated by the polymerasechain reaction (PCR), and fragments generated by any of ligation,scission, endonuclease action, and exonuclease action. Nucleic acidmolecules can be composed of monomers that are naturally-occurringnucleotides (such as DNA and RNA), or analogs of naturally-occurringnucleotides (e.g., α-enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Modified nucleotides can havealterations in sugar moieties and/or in pyrimidine or purine basemoieties. Sugar modifications include, for example, replacement of oneor more hydroxyl groups with halogens, alkyl groups, amines, and azidogroups, or sugars can be functionalized as ethers or esters. Moreover,the entire sugar moiety can be replaced with sterically andelectronically similar structures, such as aza-sugars and carbocyclicsugar analogs. Examples of modifications in a base moiety includealkylated purines and pyrimidines, acylated purines or pyrimidines, orother well-known heterocyclic substitutes. Nucleic acid monomers can belinked by phosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “expression vector” as used herein in the specification and theclaims includes nucleic acid molecules encoding a gene that is expressedin a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter. The term“promoter” refers to any DNA sequence which, when associated with astructural gene in a host yeast cell, increases, for that structuralgene, one or more of 1) transcription, 2) translation or 3) mRNAstability, compared to transcription, translation or mRNA stability(longer half-life of mRNA) in the absence of the promoter sequence,under appropriate growth conditions.

The term “oncogene” as used herein refers to genes that permit theformation and survival of malignant neoplastic cells (Bradshaw, T. K.:Mutagenesis 1, 91-97 (1986).

As used herein the term “receptor” denotes a cell-associated proteinthat binds to a bioactive molecule termed a “ligand.” This interactionmediates the effect of the ligand on the cell. Receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor). Membrane-boundreceptors are characterized by a multi-domain structure comprising anextracellular ligand-binding domain and an intracellular effector domainthat is typically involved in signal transduction. In certainmembrane-bound receptors, the extracellular ligand-binding domain andthe intracellular effector domain are located in separate polypeptidesthat comprise the complete functional receptor.

The term “hybridizing” refers to any process by which a strand ofnucleic acid binds with a complementary strand through base pairing.

The term “transfection” refers to the introduction of foreign DNA intoeukaryotic cells. Transfection may be accomplished by a variety of meansknown to the art including, e.g., calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

As used herein, the term “liposome” refers to a closed structurecomposed of lipid bilayers surrounding an internal aqueous space. Theterm “polycation” as used herein denotes a material having multiplecationic moieties, such as quaternary ammonium radicals, in the samemolecule and includes the free bases as well as thepharmaceutically-acceptable salts thereof.

A list of some of the abbreviations used throughout the specificationand the claims are listed herein below in Table 1.

TABLE 1 Abbreviations Table Abbreviation Term AE Adverse event ALTAlanine transaminase (also referred to as SGPT) ANC Absolute neutrophilcount APC Antigen Presenting Cells AST Aspartate transaminase (alsoreferred to as SGOT) BUN Blood urea nitrogen CBC Complete blood count CDCluster of differentiation CMV Cytomegalovirus CO₂ Total carbon dioxideCR Complete response CRF Case report form CTCAE Common Toxicity Criteriafor Adverse Events CTL Cytotoxic T lymphocyte DC Dendritic cell(s) DTHDelayed-type hypersensitivity ECOG PS Eastern Cooperative Oncology GroupPerformance Score ELISA Enzyme-Linked ImmunoSorbent Assay ELISPOTEnzyme-Linked ImmunoSorbent Spot ER Endoplasmic reticulum FANGbishRNA^(furin) and GMCSF Augmented Autologous Tumor Cell Vaccine FLFlt-3-Ligand GM-CSF Granulocyte Macrophage-Colony Stimulating Factor(Accession No. NM_000758) GMP Good manufacturing practice GVAX GMCSFSecreting autologous or allogenic tumor cells HLA Human LeukocyteAntigen IBC Institutional Biosafety Committee IEC Independent EthicsCommittee IL Infiltrating lymphocytes IRB Institutional Review Board LAKLymphokine-activated killer LD Longest diameter LLC Large latent complexMHC Major histocompatability complex MLR Mixed lymphocyte reaction MRMannose receptor NK Natural Killer NKT Natural Killer T cell(s) NSCLCNon small cell lung cancer PCR Polymerase chain reaction PD Progressivedisease PI Principal Investigator PR Partial response PS PerformanceStatus RECIST Response Evaluation Criteria in Solid Tumors SCLC Smallcell lung cancer SD Stable disease SLC Small latent complex STMN1Stathmin 1 TAP transporter associated with Ag processing TGF-βTransforming growth factor-β TIL Tumor infiltrating lymphocytes TNFTumor necrosis factor ULN Upper limits of normal WNL Within normallimits

Furin is a member of the subtilisin-like proprotein convertase family.The members of this family are proprotein convertases (PCs) that processlatent precursor proteins into their biologically active products.Furin, a calcium-dependent serine endoprotease, efficiently cleavesprecursor proteins at their paired basic amino acid processing sites bythe consensus sequence -Arg-X-K/Arg-Arg (RXK/RR), with —RXXR— (SEQ. IDNO: 1) constituting the minimal cleavage site. Like many otherproteases, PCs are synthesized as inactive zymogens with an N-terminalprosegment extension, which is autocatalytically removed in theendoplasmic reticulum to achieve functionality.

High levels of furin have been demonstrated in virtually all cancerlines (Furin, Accession No. NM_(—)002569). A 10-fold higher level ofTGF-β1 may be produced by human colorectal, lung cancer and melanomacells, and likely impact the immune tolerance state by a highermagnitude. Transforming growth factors betas (TGF-β) are a family ofmultifunctional proteins with well known immunosuppressive activities.The three known TGF-β ligands (TGF-β1-3, Accession Nos. NM_(—)000660,NM_(—)003238, NM_(—)003239.2, respectively) are ubiquitous in humancancers. TGF-β overexpression correlates with tumor progression and poorprognosis. Elevated TGF-β levels within the tumor microenvironment arelinked to an anergic antitumor response. The presence of furin in tumorcells likely contributes significantly to the maintenance of tumordirected TGF-β1 peripheral immune tolerance. Hence, furin knockdownrepresents a novel and attractive approach for optimizingimmunosensitization.

The incorporation of a bi-functional shRNA^(furin) in combination withhGM-CSF into an autologous cell vaccine is demonstrated herein topromote and enhance the immune response based on its effect on theafferent limb of that immune response.

Other applications for the bi-functional shRNA^(furin) include: (1)Systemic delivery via a tumor (±tumor extracellular matrix (ECM))selective decorated (targeted), stealthed bilamellar invaginatedliposome (BIV) to enhance the efferent limb of the immune response; (2)Systemic delivery via a tumor selective decorated (targeted), stealthedbilamellar invaginated liposome (BIV) to directly subvert the tumorpromoting/maintaining effects of furin target molecules including, butnot limited to, IGF-II, IGF-1R, PDGF A, and, in some tumor types,MT1-MMP; (3) Systemic delivery via a tumor selective decorated(targeted), stealthed bilamellar invaginated liposome (BIV) to directlysubvert the NOTCH/p300 pathway in putative cancer stem cells; (4)Systemic delivery via a tumor selective decorated (targeted), stealthedbilamellar invaginated liposome (BIV) to inhibit activation of toxinsassociated with anthrax, Shiga, diphtheria, tetanus, botulism and Ebolaand Marburg viruses and/or (5) Systemic and/or inhalational delivery ofa bilamellar invaginated liposome (BIV) (±decoration and reversiblemasking/stealthing) to inhibit Pseudomonas exotoxin A production as anadjunct to antibiotic therapy in patients with diseases with heightenedrisk of Pseudomonas mediated morbidity and mortality, e.g., cysticfibrosis.

A Furin-knockdown and GM-CSF-augmented (FANG) Autologous Cancer Vaccinefor Human Melanoma and Lung Cancer: FANG uniquely incorporates aproprietary bi-functional small hairpin RNA (shRNA) construct specificfor the knockdown of furin, a proprotein convertase critically involvedin the functional processing of all TGF-β isoforms. Prior work by theinventors has demonstrated the effectiveness of FANG in generatingGM-CSF expression and TGF-β1 and -β2 depletion in human cancer lines.

As used herein the term “bi-functional” refers to a shRNA having twomechanistic pathways of action, that of the siRNA and that of the miRNA.The term “traditional” shRNA refers to a DNA transcription derived RNAacting by the siRNA mechanism of action. The term “doublet” shRNA refersto two shRNAs, each acting against the expression of two different genesbut in the “traditional” siRNA mode.

Overcoming immune tolerance with cancer vaccines is a promising butdifficult quest. The prevailing hypotheses for immune tolerance, basedprimarily on animal studies, include the low immunogenicity of the tumorcells, the lack of appropriate presentation by professional antigenpresenting cells, immune selection of antigen-loss tumor variants, tumorinduced immunosuppression, and tumor-induced privileged site [1].Nevertheless, recent clinical trials that are based ontransgene-expressing whole cancer cell vaccines have yielded promisingresults [2-5]. Whole cancer cell vaccines can potentially elicitbroad-based, polyvalent immune responses to both defined and undefinedtumor antigens, thereby addressing the possibility of tumor resistancethrough downregulation and/or selection for antigen-loss variants [6,7].

Dranoff and Jaffee have shown in animal models [8], that tumor cellsgenetically modified to secrete GM-CSF, as compared to other cytokines,consistently demonstrated the most potent induction of anti-tumorimmunity. When integrated as a cytokine transgene, GM-CSF enhancespresentation of cancer vaccine peptides, tumor cell lysates, or wholetumor cells from either autologous or established allogeneic tumor celllines [9]. GM-CSF induces the differentiation of hematopoieticprecursors into professional antigen presenting (APC) dendritic cells(DC) and attracts them to the site of vaccination [8, 10]. GM-CSF alsofunctions as an adjuvant for the DC maturation and activationalprocesses of tumor antigen capture, process and presentation,upregulates their expression of costimulatory molecules, and theirability to migrate to secondary lymphoid tissues for activation of CD4+,CD8+ T cells, CD1d restricted invariant natural killer T (NKT) cells,and antibody producing B cells [11].

Recently, Hodi [12] reported that GVAX vaccination, followed by periodicinfusions of anti-CTLA-4 antibodies to modulate effector and Tregulatory cell functions, can generate clinically meaningful antitumorimmunity in a majority of metastatic melanoma patients. These findingsare consistent with the thesis that vaccination with a GM-CSF-augmentedautologous cancer vaccine can successfully generate an immune mediatedtumor destruction, particularly when coupled with an adjuvant treatmentthat depletes FoxP3+ Tregs activity, enhances tumor expression of MHCclass I A chain (MICA) thereby activating natural killer (NK) and Tcells, and enhances central memory T-cell CD4+ and CD8+ response.

TGF-β Knockdown: Transforming growth factors beta (TGF-β) are a familyof multifunctional proteins with well known immunosuppressive activities[13]. The three known TGF-β ligands (TGF-131, 132, and (33) areubiquitous in human cancers. TGF-β overexpression correlates with tumorprogression and poor prognosis [14, 15]. Elevated TGF-β levels withinthe tumor microenvironment are linked to an anergic antitumor response[14, 16-21]. TGF-β inhibits GM-CSF induced maturation of DCs [22] andtheir expression of MHC class II and co-stimulatory molecules [23].Ardeshna [24] showed that lipopolysaccharide (LPS)-induced maturation ofmonocyte-derived DCs involved activation of p38 stress-activated proteinkinase (p38SAPK), extracellular signal-regulated protein kinase (ERK),phosphoinositide 3-OH— kinase (PI3 kinase)/Akt, and nuclear factor(NF)-κB pathways. GM-CSF can exert parallel activities of stimulatingmyeloid hematopoietic cell and leukemia cell line proliferation throughrapid, transient phosphorylation of MAP kinase 1/2 and ERK 1/2, whereasTGF-β turns off GM-CSF-induced ERK signaling via PI3-kinase-Akt pathwayinhibition [25].

At the efferent level, antigen presentation by immature DCs contributesto T cell anergy [26]. TGF-β similarly inhibits macrophage activation[27] and their antigen presenting function [28, 29]. TGF-β inhibits theactivation of cytotoxic T cells by impairing high affinity IL-2 receptorexpression and function [30, 31]. TGF-β2 also converts naïve T cells toTreg cells by induction of the transcription factor FOXP3 [32], withemergence of Treg leading to the shutdown of immune activation [33].According to Polak [34], tolerogenic DCs and suppressor T lymphocyteswere present in all stages of melanoma. These immune cell typesexpressed TGF-β receptor I, and tolerogenic activity was dependent onTGF-β1 or -β2 binding.

At the innate immune response level, TGF-β is antagonistic on NK cellsand down-regulates lymphokine activated killer (LAK) cell induction andproliferation [30, 35-39]. Penafuerte [40] recently showed thattumor-secreted TGF-β suppressed GM-CSF+IL2 (GIFT2) mediatedimmunosensitization of NK cells in the immunocompetent B16 melanomamodel. In vivo blockade of B16 production of TGF-β improved survivalotherwise compromised by the growth of non-GIFT2 expressing bystandertumors. These findings further validate the negative impact of TGF-β onGM-CSF-mediated immune activation in vivo, and by extension, support therationale of depleting TGF-β secretion in GM-CSF-based cancer cellvaccines.

Trials conducted by the present inventors utilizing a tumor cell vaccinewith TGF-β2 knockdown activity (Belagenpumatucel-L) in patients withnon-small cell lung cancer demonstrated acceptable safety, and adose-related survival improvement in response to randomized controlpatients and historical experience. The two-year survival for the latestage (IIIB/IV) patients was 52% for patients who received >2.5×10⁷cells/injection, which compares favorably with similar patienthistorical data of less than 10% survival at 2 years. The study patientsalso displayed significantly elevated cytokine production (IFN-γ,p=0.006; IL-6, p=0.004; IL4, p=0.007) and antibody titers to vaccine HLAantigens (p=0.014), suggesting an immune activating outcome. [41].

TGF-β-knockdown and GM-CSF Expressive Cancer Cell Vaccine (TAG): Thirtysix patients were harvested for TAG vaccine. GM-CSF expression andTGF-β2 knockdown met product release criteria. Three (allgastrointestinal tumors with luminal access) had bacterial contaminantsand could not be released. One had insufficient cells. Nineteen advancedrefractory cancer patients were treated [42-44]. No Grade 3 toxiceffects related to therapy were observed. Eleven of 17 (65%) evaluablepatients maintained stable disease for at least 3 months. Thus the TAGvaccine appears to be safe and has evidence of clinical efficacy.

A potential limitation of TAG vaccine, however, is the restrictedspecificity for TGF-β2, given that all three known isoforms of TGF-βligand (TGF-β1, -β2, and -β3) are ubiquitously produced in humancancers. In particular, up to a 10-fold higher level of TGF-β1 may beproduced by human colorectal, lung cancer, and melanoma cells. Thetolerogenic role of TGF-β1 in antigen presenting dendritic cells (DC)and T regulatory cells (Treg) is well established, and this activity isnot impacted by TGF-β2 antisense treatment.

Furin: All mature isoforms of TGF-β require limited proteolytic cleavagefor proper activity. The essential function of proteolytic activation ofTGF-β is mediated by furin. Furin is a member of the subtilisin-likeproprotein convertase family. The members of this family are proproteinconvertases (PCs) that process latent precursor proteins into theirbiologically active products. Furin, a calcium-dependent serineendoprotease, efficiently cleaves precursor proteins at their pairedbasic amino acid processing sites by the consensus sequence-Arg-X-K/Arg-Arg (RXK/RR), with —RXXR— (SEQ. ID NO: 1) constituting theminimal cleavage site [53]. Like many other proteases, PCs aresynthesized as inactive zymogens with an N-terminal prosegmentextension, which is autocatalytically removed in the endoplasmicreticulum to achieve functionality [52].

Furin is best known for the functional activation of TGF-β withcorresponding immunoregulatory ramifications [54, 55]. Apart from thepreviously described immunosuppressive activities of tumor secretedTGF-β, conditional deletion of endogenous-expressing furin in Tlymphocytes was found to allow for normal T-cell development, butimpaired the function of regulatory and effector T cells, which producedless TGF-β1. Furin-deficient Tregs were less protective in a T-celltransfer colitis model and failed to induce Foxp3 in normal T cells.Additionally, furin-deficient effector cells were inherently over-activeand were resistant to suppressive activity of wild-type Treg cells. InAPCs, cytotoxic T lymphocyte-sensitive epitopes in the trans-Golgicompartment were processed by furin and the less frequented TAPindependent pathway [56]. Thus furin expression by T cells appears to beindispensable in maintaining peripheral tolerance, which is due, atleast in part, to its non-redundant, essential function in regulatingTGF-β1 production.

High levels of furin have been demonstrated in virtually all cancerlines [45-52]. The present inventors and others have found that up to a10-fold higher level of TGF-β1 may be produced by human colorectal, lungcancer, and melanoma cells, and likely impact the immune tolerance stateby a higher magnitude [34, 57, 58]. The presence of furin in tumor cellslikely contributes significantly to the maintenance of tumor directedTGF-β1 peripheral immune tolerance [54]. Hence furin knockdownrepresents a novel and attractive approach for optimizingimmunosensitization.

FANG (furin shRNA and GMCSF) vaccine: The present inventors constructedthe next generation vaccine termed FANG. The novelty of the FANG vaccinelies in the combined approach of depleting multiple immunosuppressiveTGF-β isoforms by furin knockdown, in order to maximize the immuneenhancing effects of the incorporated GM-CSF transgene on autologoustumor antigen sensitization.

All mature isoforms of TGF-β require proteolytic activation by furin.The feasibility of achieving concomitant depletion of multiple TGF-βisoform activity in several cancer cell lines (H460, CCL-247, CRL-1585,U87) was determined using furin-knockdown and the present inventors havesuccessfully completed GMP manufacturing of FANG vaccine in 9 cancerpatients (breast—1; colon—2; melanoma—4; gallbladder—1; NSCLC—1).Assessment of GMCSF expression and TGF-β1 and -β2 knockdown is shown inFIGS. 1A-1C.

The capability of FANG to knockdown both TGF-β1 and -β2 is supported byfindings in the first 9 patients (FIGS. 1A and 1B) who underwent vaccineconstruction. All 9 vaccine preparations demonstrated significantlyelevated levels of GM-CSF (80-1870 pg/ml at day 4 of culture, median of739 pg/ml). All 9 patients demonstrated >50% reductions of TGF-β2, and 6of 7 patients with >100 pg of endogenous TGF-β1 production alsodemonstrated >50% reduction of this cytokine. The expanded targeteffectiveness of FANG is best demonstrated in one patient (NSCLC) whohad adequate tumor tissue to generate both TAG (TAG-004A) and FANG(FANG-004) versions of autologous vaccine, TGF-β1 (as well as TGF-β2)was depleted to below detectable levels using the FANG preparation(FANG-004) from an initial concentration of 1840 pg/ml whereas this highlevel of TGF-β1 was unchanged with the TAG preparation (TAG-004) albeitwith the expected depletion of TGF-β2. These findings support thepotential advantage of the FANG vaccine preparation.

Validation of bioactivity of personalized cGMP FANG vaccines: Genemodification will be achieved by the use of a plasmid vector encodingfor GM-CSF and a bi-functional short hairpin (bi-sh) RNA optimized forfurin knockdown. Cancer patient autologous FANG vaccine has already beengenerated under cGMP conditions for clinical trial of patients withadvanced solid cancers. GM-CSF and TGF-β1, -β2, and -β3 mRNA and proteinexpression were measured as part of the quality assurance process.Cytokine bioactivity following FANG modification was determined bygrowth outcome in a GM-CSF and TGF-β dependent cell line utilized by thepresent inventors in previous studies. Processed vaccine will undergoproteogenomic screening to verify antigenic integrity following FANGmodification.

To characterize the augmenting effect of CTLA-4 blockade: Given thatFANG immunization only impacts the afferent immunosensitization process,additional approaches that promote tumor-specific immune effectorresponses may further promote antitumor outcome. Disrupting Tregsuppression and/or enhancing T effectors (Teff) by blockade of thecytotoxic T lymphocyte-4 (CTLA-4) function may enhance the likelihood ofclinical success of the FANG vaccine.

The FANG approach was supported by the findings from 9 patientautologous vaccines, which consistently demonstrated TGF-β1 and TGF-β2reductions and elevated GM-CSF levels (FIGS. 1A to 1C). Both TGF-β1 andTGF-β2 activity by specific immunoassay was also demonstrated to besignificantly reduced in these cancer lines, confirming the effect offurin blockade on TGF-β isoform expression. The inventors validated theapplicability of siRNA-mediated furin-knockdown for inhibiting TGF-βisoform expression. Prospective siRNA targeting sites (FIG. 2) in thefurin mRNA sequence were determined by the published recommendations ofTuschl and colleagues and the additional selection parameters thatintegrated BLAST searches of the human and mouse genome databases(http:jura.wi.mit.edu/bioc/siRNAext). siRNAs targeting eligibletranslated and 3′UTRs sites (FIG. 2) were tested.

Following lipofection of CCL-247, CRL-1585 U87 and H460 cells, each ofthe 6 siRNA^(furin) constructs was shown to markedly reduce TGF-β1 andTGF-β2 levels in culture supernatants without adversely affecting cellsurvival. Thus siRNA-mediated furin knockdown is effective for thedepletion of TGF-β1 and -β2 isoforms.

Design and construction of FANG: A “bi-functional” vector was used thatincorporates both siRNA and miRNA-like functional components foroptimizing gene knockdown [61]. The siRNA component is encoded as ahairpin and encompasses complete matching sequences of the passenger andguide strands. Following cleavage of the passenger strand by theArgonaute-2 (Ago 2) of the RNA-induced silencing complex (RISC), anendonuclease with RNase H like activity, the guide strand binds to andcleaves the complementary target mRNA sequence (cleavage-dependentprocess). In distinction, the miRNA-like component of the“bi-functional” vector incorporates mismatches between the passenger andguide strands within the encoding shRNA hairpin in order to achievelower thermodynamic stability. This configuration allows the passengerstrand to dissociate from RISC without cleavage (cleavage-independentprocess) independent of Ago 2 [62, 63], and the miRNA guide component todownregulate its target through translational repression, mRNAdegradation, and sequestration of the partially complementary targetmRNA in the cytoplasmic processing bodies (P-body).

The inventors have previously demonstrated the enhanced effectiveness ofa bi-functional shRNA to knockdown stathmin (STMN1; oncoprotein 18), aprotein that regulates rapid microtubule remodeling of the cytoskeletonand found to be upregulated in a high proportion of patients with solidcancers [64]. The bi-functional shRNA construct achieved effectiveknockdown against STMN-1 resulting in a 5-log dose enhanced potency oftumor cell killing as compared with siRNA oligonucleotides directedagainst the same gene target.

A similarly designed bi-functional shRNA was used to effect furinknockdown. The bi-functional shRNA^(furin) consists of two stem-loopstructures with a miR-30a backbone; the first stem-loop structure hascomplete complementary guiding strand and passenger strand, while thesecond stem-loop structure has two by mismatches at positions 11 and 12of the passenger strand. The inventors adopted a strategy of using asingle targeted site for both cleavage and sequestration processes. Theencoding shRNAs are proposed to allow mature shRNA to be loaded ontomore than one type of RISC [65]. The inventors focused on a single sitesince multi-site targeting may increase the chance for “seed sequence”induced off-target effects [66].

The two stem-loop structure was put together with 10 pieces ofcomplementing and interconnecting oligonucleotides through DNA ligation.Orientation of the inserted DNA was screened by PCR primer pairsdesigned to screen for the shRNA insert and orientation. Positive cloneswere selected and sequence confirmed at SeqWright, Inc. (Houston, Tex.).Based on siRNA findings, three bi-functional shRNA's were constructed.The optimal targeting sequence was identified.

The FANG construct has a single mammalian promoter (CMV) that drives theentire cassette, with an intervening 2A ribosomal skip peptide betweenthe GM-CSF and the furin bi-functional shRNA transcripts, followed by arabbit polyA tail. There is a stop codon at the end of the GM-CSFtranscript.

Insertion of picornaviral 2A sequences into mRNAs causes ribosomes toskip formation of a peptide bond at the junction of the 2A anddownstream sequences, leading to the production of two proteins from asingle open reading frame [67]. The inventors found that the 2A linkerto be effective for generating approximately equal levels of GM-CSF andanti-TGF-β transcripts with the TAG vaccine, and elected to use the samedesign for FANG.

cGMP FANG vaccines: Cancer patient autologous FANG vaccines weregenerated under cGMP conditions for use in clinical trials. GM-CSF andTGF-β1, -β2, and -β3 mRNA and protein expression were measured beforeand after FANG modification, and cytokine bioactivity determined bygrowth outcome on a GM-CSF and TGF-β dependent human cell line we havepreviously characterized. Each patient's processed vaccine will undergoproteogenomic screening to verify antigenic integrity following FANGmodification.

cGMP production of FANG: FANG vaccine was generated by plasmid vectorelectroporation of established human cell lines. The selected FANGplasmid vector represents a construct containing the furin shRNA thathas been prevalidated for optimal TGF-β downregulation.

Quantification of GM-CSF and TGF-β expressions: GM-CSF and TGF-β1 and-β2 expression was determined by cytokine specific colorimetric assay[68].

Validation of bioactivity: GM-CSF-induced proliferative activity similarto that of myeloid hematopoietic cells has been observed in myeloidleukemia cell lines, as mediated by the rapid and transientphosphorylation of MAP kinase 1/2 and ERK 1/2. By contrast, TGF-β turnsoff GM-CSF-mediated ERK signaling by inhibition of the PI3-kinase-Aktpathway [25]. The growth regulatory effects of GM-CSF and TGF-β onmyeloid leukemic cells were used as an in vitro surrogate model tovalidate cytokine bioactivity in prepared FANG vaccine culturesupernatants.

Cytokine activities in the FANG (or control-transfected) vaccine culturesupernatants were validated by co-culture studies with erythroleukemicCD34+ TF-1a cells [69] and, if necessary, confirmed with thebiphenotypic B myelomonocytic leukemic CD10+ CD15+ MV4-11 cells [70](ATCC, Rockville, Md.). Both of these cell lines have been shown respondto the positive proliferative effects of GM-CSF and the negativeinhibitory activity of TGF-β at ng/ml amounts [25]. Proliferativeactivity will be determined by Easycount Viasure assay (Immunicon) andMTT assay [68].

Phenotypic profile analysis of FANG modification: Furin knockdown likelyimpacts the expression of other protein substrates with the targetsequence in addition to TGF-β downregulation [51]. The antigenic profileof the FANG-processed autologous vaccines were determined from cancerpatients, in the event that this information may be useful towards theunderstanding any differential clinical outcome in vaccinated patients.

High throughput genetic profiling was used to develop individualizedtherapeutics for cancer patients. High throughput, gene expression arrayanalysis was carried out to compare the differential gene expressionprofile of FANG-transfected vs. control vector-transfected cancer cells.

Differentially labeled FANG and control preparations are combined andfractionated by high performance liquid chromatography (Dionex), using astrong cation exchange column (SCX) and a 2^(nd) dimension RP nanocolumn. The fractions are spotted onto Opti-TOF™ LC/MALDI Insert (123×81mm) plates (Applied Biosystems) in preparation for mass spectrometryanalysis using the Applied Biosystems 4800 MALDI TOF/TOF™ Analyzer. Bothprotein and gene expression data were then evaluated by the GeneGo,MetaCore software suite.

Proteogenomic analysis was carried out for the purpose of determiningthe antigen repertoire of the autologous cancer vaccine before and afterFANG process. In addition to the validation of furin knockdown,particular attention was focused on 1) baseline and differentialexpression of furin-substrate proteins; 2) expression of landmarktumor-associated antigens (TAAs; such as gp100, Mart1, MAGE-1,tyrosinase, for melanoma; MAGE-3, MUC-1 for non-small cell lung cancer)[71, 72] and other reported TAAs; 3) HLA antigens and co-stimulatorymolecules (CD80/86) expression; 4) proteins unrelated to the abovecategories that are differentially expressed by 2-fold or higherfollowing FANG transfection.

In FIG. 3 the overall survival for Cohort 1 versus Cohorts 2 and 3 foradvanced-stage patients (n=61; P=0.0186) is shown. A schematic diagramof GM-CSF-TGF-β2 antisense plasmid is represented by FIG. 4. Theexpression of GM-CSF in NCI-H-460 Squamous Cell and NCI-H-520, LargeCell (NSCLC) containing the pUMVC3-GM-CSF-2A-TGF-β2 antisense vector, invitro is depicted in FIG. 5.

Data presented in FIG. 6 shows the reduction in TGF-β2 levels inNCI-H-460 Squamous Cell and NCI-H-520, Large Cell (NSCLC) with thepUMVC3-GM-CSF-2A-TGF-β2 antisense vector. FIG. 7 shows that a 251 basepair probe specifically detects the GM-CSF-2A-TGF-β2 transcriptexpressed in vitro in NCI-H-460 and NCI-H-520 cells (lanes 6 and 10).FIGS. 8 and 9 shows the GM-CSF and TGF-β2 expression in TAG vaccines,respectively.

FIG. 10A shows the expression TGF-β1 in human cancer lines followingsiRNA^(furin) knockdown. Similar expression profile for TGF-β2 is shownin FIG. 10B. FIG. 11 shows the plasmid construct of FANG

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skilled in the art recognizethe modified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

-   U.S. Pat. No. 7,763,461: Antitumor vaccination using allogeneic    tumor cells expressing alpha (1,3)-galactosyl transferase.-   U.S. Patent Application No. 20040242518: Influenza therapeutic.-   1. Murphy, K., Travers, P., Walport, M., ed. Janeway's    Immunobiology. 7th ed. 2008, Garland Science New York. 674-687.-   2. Fakhrai, H., et al., Phase I clinical trial of a TGF-beta    antisense-modified tumor cell vaccine in patients with advanced    glioma. Cancer Gene Ther, 2006. 13(12): p. 1052-60.-   3. Nemunaitis, J., GVAX (GMCSF gene modified tumor vaccine) in    advanced stage non small cell lung cancer. J Control Release, 2003.    91(1-2): p. 225-31.-   4. Nemunaitis, J., et al., Phase ½ trial of autologous tumor mixed    with an allogeneic GVAX vaccine in advanced-stage non-small-cell    lung cancer. Cancer Gene Ther, 2006. 13(6): p. 555-62.-   5. Nemunaitis, J. and J. Nemunaitis, A review of vaccine clinical    trials for non-small cell lung cancer. Expert Opin Biol Ther, 2007.    7(1): p. 89-102.-   6. Ahmad, M., R. C. Rees, and S. A. Ali, Escape from immunotherapy:    possible mechanisms that influence tumor regression/progression.    Cancer Immunol Immunother, 2004. 53(10): p. 844-54.-   7. Hege, K. M., K. Jooss, and D. Pardoll, GM-CSF gene-modified    cancer cell immunotherapies: of mice and men. Int Rev Immunol, 2006.    25(5-6): p. 321-52.-   8. Dranoff, G., et al., Vaccination with irradiated tumor cells    engineered to secrete murine granulocyte-macrophage    colony-stimulating factor stimulates potent, specific, and    long-lasting anti-tumor immunity. Proc Natl Acad Sci USA, 1993.    90(8): p. 3539-43.-   9. Hege, K. M. and D. P. Carbone, Lung cancer vaccines and gene    therapy. Lung Cancer, 2003. 41 Suppl 1: p. S103-13.-   10. Huang, A. Y., et al., Role of bone marrow-derived cells in    presenting MHC class I-restricted tumor antigens. Science, 1994.    264(5161): p. 961-5.-   11. Banchereau, J., et al., Immunobiology of dendritic cells. Annu    Rev Immunol, 2000. 18: p. 767-811.-   12. Hodi, F. S., et al., Immunologic and clinical effects of    antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in    previously vaccinated cancer patients. Proc Natl Acad Sci USA, 2008.    105(8): p. 3005-10.-   13. Wick, W., U. Naumann, and M. Weller, Transforming growth    factor-beta: a molecular target for the future therapy of    glioblastoma. Curr Pharm Des, 2006. 12(3): p. 341-9.-   14. Bierie, B. and H. L. Moses, Tumour microenvironment: TGFbeta:    the molecular Jekyll and Hyde of cancer. Nat Rev Cancer, 2006.    6(7): p. 506-20.-   15. Levy, L. and C. S. Hill, Alterations in components of the    TGF-beta superfamily signaling pathways in human cancer. Cytokine    Growth Factor Rev, 2006. 17(1-2): p. 41-58.-   16. Sporn, M. B., et al., Transforming growth factor-beta:    biological function and chemical structure. Science, 1986.    233(4763): p. 532-4.-   17. Massague, J., The TGF-beta family of growth and differentiation    factors. Cell, 1987. 49(4): p. 437-8.-   18. Bodmer, S., et al., Immunosuppression and transforming growth    factor-beta in glioblastoma. Preferential production of transforming    growth factor-beta 2. J Immunol, 1989. 143(10): p. 3222-9.-   19. Border, W. A. and E. Ruoslahti, Transforming growth factor-beta    in disease: the dark side of tissue repair. J Clin Invest, 1992.    90(1): p. 1-7.-   20. Chen, T. C., et al., TGF-B2 and soluble p55 TNFR modulate VCAM-1    expression in glioma cells and brain derived endothelial cells. J    Neuroimmunol, 1997. 73(1-2): p. 155-61.-   21. Li, M. O., et al., Transforming growth factor-beta regulation of    immune responses. Annu Rev Immunol, 2006. 24: p. 99-146.-   22. Yamaguchi, Y., et al., Contrasting effects of TGF-beta 1 and    TNF-alpha on the development of dendritic cells from progenitors in    mouse bone marrow. Stem Cells, 1997. 15(2): p. 144-53.-   23. Geissmann, F., et al., TGF-beta 1 prevents the noncognate    maturation of human dendritic Langerhans cells. J Immunol, 1999.    162(8): p. 4567-75.-   24. Ardeshna, K. M., et al., The PI3 kinase, p38 SAP kinase, and    NF-kappaB signal transduction pathways are involved in the survival    and maturation of lipopolysaccharide-stimulated human    monocyte-derived dendritic cells. Blood, 2000. 96(3): p. 1039-46.-   25. Montenegro, D. E., et al., TGFbeta inhibits GM-CSF-induced    phosphorylation of ERK and MEK in human myeloid leukaemia cell lines    via inhibition of phosphatidylinositol 3-kinase (PI3-k). Cell    Prolif, 2009. 42(1): p. 1-9.-   26. Steinman, R. M., et al., Dendritic cell function in vivo during    the steady state: a role in peripheral tolerance. Ann N Y Acad    Sci, 2003. 987: p. 15-25.-   27. Ashcroft, G. S., Bidirectional regulation of macrophage function    by TGF-beta. Microbes Infect, 1999. 1(15): p. 1275-82.-   28. Du, C. and S. Sriram, Mechanism of inhibition of LPS-induced    IL-12p40 production by IL-10 and TGF-beta in ANA-1 cells. J Leukoc    Biol, 1998. 64(1): p. 92-7.-   29. Takeuchi, M., P. Alard, and J. W. Streilein, TGF-beta promotes    immune deviation by altering accessory signals of antigen-presenting    cells. J Immunol, 1998. 160(4): p. 1589-97.-   30. Ruffini, P. A., et al., Factors, including transforming growth    factor beta, released in the glioblastoma residual cavity, impair    activity of adherent lymphokine-activated killer cells. Cancer    Immunol Immunother, 1993. 36(6): p. 409-16.-   31. Fakhrai, H., et al., Eradication of established intracranial rat    gliomas by transforming growth factor beta antisense gene therapy.    Proc Natl Acad Sci USA, 1996. 93(7): p. 2909-14.-   32. Fantini, M. C., et al., Cutting edge: TGF-beta induces a    regulatory phenotype in CD4+CD25− T cells through Foxp3 induction    and down-regulation of Smad7. J Immunol, 2004. 172(9): p. 5149-53.-   33. Thomas, D. A. and J. Massague, TGF-beta directly targets    cytotoxic T cell functions during tumor evasion of immune    surveillance. Cancer Cell, 2005. 8(5): p. 369-80.-   34. Polak, M. E., et al., Mechanisms of local immunosuppression in    cutaneous melanoma. Br J Cancer, 2007. 96(12): p. 1879-87.-   35. Rook, A. H., et al., Effects of transforming growth factor beta    on the functions of natural killer cells: depressed cytolytic    activity and blunting of interferon responsiveness. J Immunol, 1986.    136(10): p. 3916-20.-   36. Kasid, A., G. I. Bell, and E. P. Director, Effects of    transforming growth factor-beta on human lymphokine-activated killer    cell precursors. Autocrine inhibition of cellular proliferation and    differentiation to immune killer cells. J Immunol, 1988. 141(2): p.    690-8.-   37. Tsunawaki, S., et al., Deactivation of macrophages by    transforming growth factor-beta. Nature, 1988. 334(6179): p. 260-2.-   38. Hirte, H. and D. A. Clark, Generation of lymphokine-activated    killer cells in human ovarian carcinoma ascitic fluid:    identification of transforming growth factor-beta as a suppressive    factor. Cancer Immunol Immunother, 1991. 32(5): p. 296-302.-   39. Naganuma, H., et al., Transforming growth factor-beta inhibits    interferon-gamma secretion by lymphokine-activated killer cells    stimulated with tumor cells. Neurol Med Chir (Tokyo), 1996.    36(11): p. 789-95.-   40. Penafuerte, C. and J. Galipeau, TGF beta secreted by B16    melanoma antagonizes cancer gene immunotherapy bystander effect.    Cancer Immunol Immunother, 2008. 57(8): p. 1197-206.-   41. Nemunaitis, J., et al., Phase II trial of Belagenpumatucel-L, a    TGF-beta2 antisense gene modified allogeneic tumor vaccine in    advanced non small cell lung cancer (NSCLC) patients. Cancer Gene    Ther, 2009. 16(8): p. 620-4.-   42. Maples PB, K. P., Oxendine I, Jay C, Yu Y, Kuhn J, Nemunaitis J,    TAG Vaccine: Autologous Tumor Vaccine Genetically Modified to    Express GM-CSF and Block Production of TGFB2. BioProcessing    Journal, 2009. 8(2).-   43. Nemunaitis, J., Kumar, P., Senzer, N., Yu, Y., Oxendine, I.,    Tong, A. W., Maples, P. B., A phase I trial of GMCSF gene-TGFbeta    antisense gene autologous tumor cell (TAG) vaccine in advanced    cancer. Mol Therapy, 2009. 17 (Suppl 1): p. 5206.-   44. Maples, P. B., et al. Autologous Tumor Cell Vaccine Genetically    Modified To Express GM-CSF and Block Expression of TGFb2 (Abstract    #553). in The Twelfth Annual Meeting of the American Society of Gene    Therapy. 2009. San Diego, Calif.-   45. Page, R. E., et al., Increased expression of the pro-protein    convertase furin predicts decreased survival in ovarian cancer. Cell    Oncol, 2007. 29(4): p. 289-99.-   46. Schalken, J. A., et al., fur gene expression as a discriminating    marker for small cell and nonsmall cell lung carcinomas. J Clin    Invest, 1987. 80(6): p. 1545-9.-   47. Mbikay, M., et al., Comparative analysis of expression of the    proprotein convertases furin, PACE4, PC1 and PC2 in human lung    tumours. Br J Cancer, 1997. 75(10): p. 1509-14.-   48. Cheng, M., et al., Pro-protein convertase gene expression in    human breast cancer. Int Cancer, 1997. 71(6): p. 966-71.-   49. Bassi, D. E., H. Mahloogi, and A. J. Klein-Szanto, The    proprotein convertases furin and PACE4 play a significant role in    tumor progression. Mol Carcinog, 2000. 28(2): p. 63-9.-   50. Bassi, D. E., et al., Elevated furin expression in aggressive    human head and neck tumors and tumor cell lines. Mol Carcinog, 2001.    31(4): p. 224-32.-   51. Lopez de Cicco, R., et al., Human carcinoma cell growth and    invasiveness is impaired by the propeptide of the ubiquitous    proprotein convertase furin. Cancer Res, 2005. 65(10): p. 4162-71.-   52. Khatib, A. M., et al., Proprotein convertases in tumor    progression and malignancy: novel targets in cancer therapy. Am J    Pathol, 2002. 160(6): p. 1921-35.-   53. Thomas, G., Furin at the cutting edge: from protein traffic to    embryogenesis and disease. Nat Rev Mol Cell Biol, 2002. 3(10): p.    753-66.-   54. Pesu, M., et al., T-cell-expressed proprotein convertase furin    is essential for maintenance of peripheral immune tolerance.    Nature, 2008. 455(7210): p. 246-50.-   55. Pesu, M., et al., Proprotein convertase furin is preferentially    expressed in T helper 1 cells and regulates interferon gamma.    Blood, 2006. 108(3): p. 983-5.-   56. Lu, J., et al., TAP-independent presentation of CTL epitopes by    Trojan antigens. J Immunol, 2001. 166(12): p. 7063-71.-   57. Fogel-Petrovic, M., et al., Physiological concentrations of    transforming growth factor beta1 selectively inhibit human dendritic    cell function. Int Immunopharmacol, 2007. 7(14): p. 1924-33.-   58. Bommireddy, R. and T. Doetschman, TGFbeta1 and Treg cells:    alliance for tolerance. Trends Mol Med, 2007. 13(11): p. 492-501.-   59. Henrich, S., et al., The crystal structure of the proprotein    processing proteinase furin explains its stringent specificity. Nat    Struct Biol, 2003. 10(7): p. 520-6.-   60. Pearton, D. J., et al., Proprotein convertase expression and    localization in epidermis: evidence for multiple roles and    substrates. Exp Dermatol, 2001. 10(3): p. 193-203.-   61. Rao, D., Maples, P. B., Senzer, N., Kumar, P., Wang, Z.,    papper, B. O., Yu, Y., Haddock, C., Tong, A., Nemunaitis, J.,    Bi-functional shRNA: A novel approach of RNA interference.    (submitted), 2009.-   62. Matranga, C., et al., Passenger-strand cleavage facilitates    assembly of siRNA into Ago2-containing RNAi enzyme complexes.    Cell, 2005. 123(4): p. 607-20.-   63. Leuschner, P. J., et al., Cleavage of the siRNA passenger strand    during RISC assembly in human cells. EMBO Rep, 2006. 7(3): p.    314-20.-   64. Rana, S., et al., Stathmin 1: a novel therapeutic target for    anticancer activity. Expert Rev Anticancer Ther, 2008. 8(9): p.    1461-70.-   65. Azuma-Mukai, A., et al., Characterization of endogenous human    Argonautes and their miRNA partners in RNA silencing. Proc Natl Acad    Sci USA, 2008. 105(23): p. 7964-9.-   66. Jackson, S. A., S. Koduvayur, and S. A. Woodson, Self-splicing    of a group I intron reveals partitioning of native and misfolded RNA    populations in yeast. RNA, 2006. 12(12): p. 2149-59.-   67. Funston, G. M., et al., Expression of heterologous genes in    oncolytic adenoviruses using picornaviral 2A sequences that trigger    ribosome skipping. J Gen Virol, 2008. 89(Pt 2): p. 389-96.-   68. Tong, A. W., et al., Intratumoral injection of INGN 241, a    nonreplicating adenovector expressing the melanoma-differentiation    associated gene-7 (mda-7/IL24): biologic outcome in advanced cancer    patients. Mol Ther, 2005. 11(1): p. 160-72.-   69. Hu, X., et al., Characterization of a unique factor-independent    variant derived from human factor-dependent TF-1 cells: a    transformed event. Leuk Res, 1998. 22(9): p. 817-26.-   70. Santoli, D., et al., Synergistic and antagonistic effects of    recombinant human interleukin (IL) 3, IL-1 alpha, granulocyte and    macrophage colony-stimulating factors (G-CSF and M-CSF) on the    growth of GM-CSF-dependent leukemic cell lines. J Immunol, 1987.    139(10): p. 3348-54.-   71. Romero, P., Current state of vaccine therapies in non-small-cell    lung cancer. Clin Lung Cancer, 2008. 9 Suppl 1: p. S28-36.-   72. Robinson, J., et al., The European searchable tumour line    database. Cancer Immunol Immunother, 2009.

What is claimed is:
 1. An expression vector comprising: a first nucleicacid insert operably linked to a promoter, wherein the first insertencodes a human Granulocyte Macrophage Colony Stimulating Factor(GM-CSF) cDNA; and a second nucleic acid insert operably linked to thepromoter, wherein the second insert encodes one or more bifunctionalshort hairpin RNAs (shRNA) capable of hybridizing to one of more regionsof a mRNA transcript encoding furin, wherein at least one of the regionsis selected from base sequences 300-318, 731-740, 1967-1991, 2425-2444,2827-2851 or 2834-2852 of SEQ ID NO:2, thereby inhibiting furinexpression via RNA interference, wherein each bifunctional short hairpinRNA comprises a first stem-loop structure that comprises an siRNAcomponent and a second stem-loop structure that comprises a miRNAcomponent and wherein the shRNA incorporates siRNA (cleavage dependent)and miRNA (cleavage-independent) motifs.
 2. The expression vector ofclaim 1, wherein a nucleotide sequence encoding a picornaviral 2Aribosomal skip peptide sequence is intercalated between the first andthe second nucleic acid inserts.
 3. The expression vector of claim 1,wherein the promoter is a CMV mammalian promoter.
 4. The expressionvector of claim 3, wherein the CMV mammalian promoter contains a CMV IE5′ UTR enhancer sequence and a CMV IE Intron A.
 5. An expression vectorcomprising a bifunctional small hairpin construct specific for knockdownof furin, wherein the expression vector plasmid further comprises: anucleic acid insert operably linked to a promoter, wherein the insertencodes one or more short hairpin RNAs (shRNA) capable of hybridizing toa region of a mRNA transcript encoding furin selected from at least oneof base sequences 300-318, 731-740, 1967-1991, 2425-2444, 2827-2851 or2834-2852 of SEQ ID NO:2, thereby inhibiting furin expression via RNAinterference, wherein each bifunctional short hairpin RNA comprises afirst stem-loop structure that comprises an siRNA component and a secondstem-loop structure that comprises a miRNA component.
 6. The expressionvector of claim 5, wherein the siRNA component functions in acleavage-dependent manner and the miRNA component functions in acleavage-independent manner.
 7. A composition for treating cancercomprising a therapeutically effective amount of cells transfected withan expression vector comprising a first nucleic acid insert operablylinked to a promoter, wherein the first insert encodes GM-C SF and asecond nucleic acid insert operably linked to the promoter, wherein thesecond insert encodes one or more short hairpin RNAs (shRNA) capable ofhybridizing to one or more regions of a mRNA transcript encoding furin,wherein at least one of the regions is selected from base sequences300-318, 731-740, 1967-1991, 2425-2444, 2827-2851 or 2834-2852 of SEQ IDNO:2, thereby inhibiting furin expression via RNA interference, whereineach bifunctional short hairpin RNA comprises a first stem-loopstructure that comprises an siRNA component and a second stem-loopstructure that comprises a miRNA component.
 8. The composition of claim7, wherein the GM-CSF is human.
 9. The composition of claim 7, whereinthe siRNA component functions in a cleavage-dependent manner and themiRNA component functions in a cleavage-independent manner.
 10. Thecomposition of claim 7, wherein a nucleotide sequence encoding apicornaviral 2A ribosomal skip peptide sequence is intercalated betweenthe first and the second nucleic acid inserts.
 11. The composition ofclaim 7, where the promoter is a CMV mammalian promoter, enhancer andintron.
 12. The composition of claim 7, wherein the cells are xenograftexpanded tumor cells.
 13. The composition of claim 7, wherein thecomposition comprises 1×10⁷ cells to 2.5×10⁷ cells.
 14. The compositionof claim 7, wherein the composition further comprises a therapeuticallyeffective dose of γIFN (gamma interferon).
 15. The composition of claim14, wherein the therapeutically effective dose of γIFN is 50 or 100μg/m².